A method used to detect an antigen concentration includes forming an antibody fixed film having an antibody attached to a thin metallic film exposed to a flow path portion of a flow cell, flowing liquid containing an antigen in the flow cell, detecting the antigen attached to the antibody, i.e., a change of a refractive index of the antibody fixed film due to an antigen-antibody complex using a surface plasmon resonance sensor, and detecting a concentration of the antigen from a rate of change of the refractive index (see, for example, Non-Patent Document 1).
Accordingly, since the rate of change of the refractive index increases with the antigen concentration, the antigen concentration is measured from the rate of change.
A surface plasmon resonance (hereinafter, referred to as SPR) measurement apparatus has recently been studied as a bio sensor using light (see, for example, Patent Documents 1 and 2). In the antigen concentration measurement apparatus, a device in which a measured substance, such as an antibody, is fixed on a thin metallic film, such as gold or silver, is used as a measurement flow cell. Light is emitted from a surface opposing the antibody of the flow cell, and an incident angle at which a resonance between an evanescence wave and a surface plasmon wave occurs is measured.
FIG. 18 is a schematic block diagram showing a configuration of a conventional antigen concentration measurement apparatus. The antigen concentration measurement apparatus has a prism 1001, a light source 1002, a polarizer 1003, a focusing lens 1004, and a CCD camera 1005.
When light radiated from the light source 1002 for monochromatic light passes through the polarizer 1003, only P-polarized light passes. This P-polarized light is focused by the focusing lens 1004 and emitted to the hemispherical prism 1001. A flow cell 1000 is disposed on an upper surface of the prism 1001, and the P-polarized light is emitted from a surface opposing the surface to which a measured substance, such as an antibody, is fixed. Thus, the P-polarized light is emitted to the flow cell 1000 at an incident angle θ via the prism 1001, such that a change of intensity of reflected light from the flow cell 1000 is detected by the CCD camera 1005.
The light radiated from the light source 1002 is converted into an evanescent wave at an interface between the prism 1001 and the thin metallic film of the flow cell 1000. Meanwhile, a surface plasmon wave is generated on a surface of the thin metallic film. At an incident angle θ at which a wave number of the evanescent wave is coincident with that of the surface plasmon wave, the evanescent wave is used to excite the surface plasmon wave and a light amount measured from the reflected light is reduced.
In this case, if the intensity of the reflected light is measured by the CCD camera 1005, degradation of the reflectivity is observed at an incident angle at which the resonance between the evanescence wave and the surface plasmon wave occurs, as shown in FIG. 19. On an incident angle-reflectivity curve indicating a relationship between the incident angle and the reflectivity, this is shown as a valley with low reflectivity around the incident angle at which the resonance between the evanescence wave and the surface plasmon wave occurs.
Since the angle at which the resonance between the evanescence wave and the surface plasmon wave occurs depends on a refractive index of the measured substance contiguous to the thin metallic film of the flow cell 1000, when the measured substance such as an antibody is fixed on the thin metallic film, the refractive index of the antibody is changed due to coupling with the antigen, and the angle at which the valley is shown undergoes a slight change, which can be measured to determine an amount of the measured substance.    Patent Document 1: Japanese Patent Application, First Publication No. 2001-194298    Patent Document 2: Japanese Patent No. 3356213    Non-Patent Document 1: Milan Mrksich, George B. Sigal, and George M. Whitesides, “Surface Plasmon Resonance Permits in Situ Measurement of Protein Adsorption on Self-Assembled Monolayers of Alkanethiolates on Gold,” American Chemical Society, Langmuir, 1995, 11, 4383-4385